Synthesis of Chlorodideoxynucleosides Using Tris(2,4,6-tribromophenoxy)-dichlorophosphorane (BDCP)

Abstract

5′-Chloro-5′-deoxy-N,3′-O-dibenzoylthymidine (3a), 5′-chloro-5′-deoxy-N⁴, 3′-O-dibenzoyldeoxycytidine(3b), 5′-chloro-5′-deoxy-N⁶,3′-O-dibenzoyldeoxyadenosine(3c), N-benzoyl-1-(3-chloro-2,3-dideoxy-5-O-trityl-ß-D-xylofuranosyl)thymine (5a) and N⁶-benzoyl-9-(3-chloro-2,3-dideoxy-5-O-trityl-ß-D-xylofuranosyl)adenine (5b) have been synthesized in very high yields using a new efficient reagent, tris(2,4,6-tribrom-ophenoxy)dichlorophosphorane (BDCP). The reaction time was greatly reduced to 5–8 min. NOE data suggested an inversion of configuration at C₃-position and thus an SN2 mechanism has been proposed for the chlorination reaction.

     

Purification of 5′-O-Trityl-on Oligoribonucleotides. Investigation of Phosphate Migration During Purification and Detritylation.

Abstract

Synthetic oligoribonucleotides (RNA) are efficiently prepared with 2′-O-tert-butyldimethylsilyl nucleoside 3′-O-phosphoramidites with labile base-protection; A_dmf or A_Pac, G_dmf, C_ibu, U. After cleavage from the polystyrene support, the exocyclic amine protecting groups are removed with conc. NH₄OH: ethanol/3:1 by heating at 55°C for 3–5 h. The 2′-O- silyl protecting groups are removed with tetra-n-butylammonium fluoride in THF or more conveniently with neat triethylamine trihydrofluoride. To gain the advantages of increased capacity on reverse phase HPLC and the convenience of cartridge based purification (OPC, Oligonucleotide Purification Cartridge), the 5′ trityl was left on the RNA as the final protecting group to be removed. The mild conditions which are effective for trityl removal are shown to preserve 3′-5′ phosphate linkage integrity in RNA. The absence of phosphate migration is demonstrated by model studies, utilizing N⁴ -isobutyryl-5′-O-DMT-3′-O-TBDMS-2′-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite) as a control monomer and digestion by 3′-5′ selective P1 nuclease and alkaline phosphatase and HPLC analysis. Oligoribonucleotides were analyzed by Microgel capillary electrophoresis, anion-exchange HPLC, and the enzymatic digest/HPLC method.

     

Chemical and Biological O-Demethylation of Rotenone Derivatives

3-O-Demethyl and 2,3-O,O-didemethyl derivatives of natural rotenone (5′β-rotenone), 5′α-rotenone, d-epirotenone (5′β-epirotenone) and 5′α-epirotenone are obtained upon reacting 5′β-rotenone or 5′β-epirotenone with two or three molar equivalents of boron tribromide followed by recyclization of the E-ring using sodium bicarbonate. 3-Methoxy-¹⁴C-5′β-rotenone is prepared in 16% yield by treating 3-O-demethyl-5′β-rotenone with methyl-¹⁴C iodide in the presence of alkali followed by epimerization of the ¹⁴C-5′β-epirotenone byproduct for increased yield of ¹⁴C-5′β-rotenone. 3-O-Demethylation is established as a detoxification mechanism for 5′β-rotenone or for one of its metabolites based on the expiration by mice and rats of 27% and 13%, respectively, of the administered radiocarbon as ¹⁴carbon dioxide.     

Synthesis of (E)-5-(2-cIOdovinyl)-3′-0-(1-Methyl-1,4-Dihydropyridyl-3 -Carbonyl)-2′-Fluoro-2′-Deoxyuridine (Ivfru-Cds) for Brain Targetted Delivery of Ivfru,an Antiviral Nucleoside

Abstract

(E)-5-(2-lodovinyl)-2′-fluoro-3′-0-(1-methyl-1,4-dihydropyridyl-3-carbonyl)-2′-deoxyuridine (11) was synthesized for future evaluation as a lipophilic, brain-selective, pyrimidine phosphorylase-resistant, antiviral agent for the treatment of Herpes simplex encephalitis (HSE). Treatment of (E)-5-(2-iodovinyl)-2′-fluoro-2′-deoxyuridine (6) with TBDMSCI in the presence of imidazole in DMF yielded the protected 5′-O-t-butyldimethylsilyl derivative (7). Subsequent reaction with nicotinoyl chloride hydrochloride in pyridine afforded (E)-5-(-2-iodovinyl)-2′-fluoro-3′-O-(3-pyridylcarbonyl)-5′-O-t-butyldimethylsily-2′-deoxyuridine (8). Deprotection of the silyl ether moiety of 8 with n-Bu₄N⁺F^? and quaternization of the resulting 3′-O-(3-pyridylcarbonyl) derivative 9 using iodomethane afforded the corresponding 1-methylpyridinium salt 10. The latter was reduced with sodium dithionite to yield (E)-5-(2-iodovinyl)-2′-fluoro-3′-O-(1-methyl-1,4-dihydropyridyl-3-carbonyl)-2′-deoxyuridine (11).     

CycloSaligenyl Pronucleotides of 5-Iodo and 5-Trifluoromethyl-1-(2-deoxy-β-D-ribofuranosyl)-2,4-difluorobenzene Mimics of Thymidine: Synthesis and Evaluation of this Pronucleotide Monophosphate Delivery System for Compounds with Potential Anticancer Activity

Abstract

A group of unnatural 1-(2-deoxy-β-D-ribofuranosyl)-2,4-difluorobenzenes possessing a 5-I or 5-CF₃ substituent, that were originally designed as thymidine mimics, were coupled via their 5′-OH group to a cyclosaligenyl (cycloSal) ring system having a variety of C-3 substituents (Me, OMe, H). The 5′-O-cycloSal-pronucleotide concept was designed to effect a thymidine kinase-bypass, thereby providing a method for the intracellular delivery and generation of the 5′-O-monophosphate for nucleosides that are poorly phosphorylated. The 5′-O-cycloSal pronucleotide phosphotriesters synthesized in this study were obtained as a 1:1 mixture of two diastereomers that differ in configuration (S _P or R _P) at the asymmetric phosphorous center. The (S _P)- and (R _P)-diastereomers for the 5′-O-3-methylcycloSal- and 5′-O-3-methoxycycloSal derivatives of 1-(2-deoxy-β-D-ribofuranosyl)-2,4-difluoro-5-iodobenzene were separated by silica gel flash column chromatography. This class of cycloSal pronucleotide compounds generally exhibited weak cytotoxic activities in a MTT assay (CC₅₀ values in the 10^?3 to 10^?4 M range), against a number of cancer cell lines (143B, 143B-LTK, EMT-6, Hela, 293), except for cyclosaligenyl-5′-O-[1′-(2,4-difluoro-5-iodophenyl)-2′-deoxy-β-D-ribofuranosyl]phosphate that was more potent (CC₅₀ values in the 10^?5 to 10^?6 M range), than the reference drug 5-iodo-2′-deoxyuridine (IUDR) which showed CC₅₀ values in the 10^?3 to 10^?5 M range.     

Efficient Synthesis of 2′-Bromo-2′-Deoxy-3′,5′-O-TPDS-pyrimidine Nucleosides by Boron Trifluoride Catalyzed Reaction of O2,2′-Anhydro-(1-β-D-Arabinofuran0Syl)pyrimidine Nucleosides with Lithium Bromide

Abstract

Efficient syntheses of 2′-bromo-2′-deoxy-3′,5′-O-TPDS-uridine (5a) and 1-(2-bromo-3,5-O-TPDS-β-D-ribofuranosyl)thymine (5b) from uridine and 1-(β-D-ribofuranosyl)thymine are described, respectively. The key step is a treatment of 3′,5′-O-TPDS-O²,2′-anhydro-1-(β-D-ardbinofuranosyl)uracil (4a) and -thymine (4b) with LiBr in the presence of BF₃-OEt₂ in 1,4-dioxane at 60°C to give 5a and 5b in 98%, and 96% yield, respectively.

     

Conformational features of 2′-O-methyl-adenosylyl-adenosine

In order to obtain information about the conformational features of a 2′-O-methylated polyribonucleotide at the nearest neighbor level, a detailed nuclear magnetic resonance study of AmpA was undertaken. AmpA was isolated from alkali hydrolysates of yeast RNA, and proton spectra were recorded at 100 MHz in the Fourier transform mode in D₂O solutions, 0.01 M, pH 5.4 and 1.5 at 25°C. ³¹P spectra were recorded at 40.48 MHz. Complete, accurate sets of nmr parameters derived for each nucleotidyl unit by simulation iteration methods. The nmr data were translated into conformational parameters for all the bonds using procedures developed in earlier studies from these laboratories. It is shown that AmpA exists in aqueous solution with a flexible molecular framework, which shows preferences for certain orientations. The ribose rings exist as a ²E ? ³E equilibrium with the —pA ribose showing a bias for the ³E pucker. The C(4′)—C(5′) bonds of both nucleotidyl units show significant preference (75–80%) to exist in gg conformation. The dominant conformer (80%) about C(5′)—O(5′) of the 5′-nucleotidyl unit is g′g′. Even though an unambiguous determination of the orientation of the 3′-phosphate group cannot be made, tentative evidence shows that it preferentially occupies g⁺ domains [O(3′)—P trans to C(3′)—C(2′)] in which the H(3′) —C(3′)—O(3′)—P(3′) dihedral angle is about 31°. There is reasonable evidence that the 2′-O-methyl preferentially occupies the domain in which the O(2′)—CH₃ bond is trans to C(2′)—C(1′). Lowering of pH to 1.5, which results in protonation of both the adenine moieties, causes destacking of AmpA. Such destacking is accompanied by small, but real, perturbations in the conformations about most of the bonds in the backbone. A detailed comparison of the solution conformations of ApA and AmpA clearly shows that 2′-O-methylation strongly influences the conformational preference about the C(3′)—O(3′) bond of the 3′-nucleotidyl unit, in addition to inducing small changes in the overall ribophosphate backbone conformational equilibria. The effect of 2′-O-methylation is such that the C(3′)—O(3′) is forced to occupy preferentially the g⁺ domain rather than the normally preferred g^? domain [O(3′)—P trans to C(3′)—C(4′)] in ApA. The data on ApA and AmpA further reveal that the extent of stacking interaction is less in AmpA compared to ApA. It is suggested that stacked species of AmpA exist as right-handed stacks where the magnitude of ω and ω′ about O(5′)—P and P—O(3′) is about 290°. The reason for the lesser degree of stacking in AmpA compared to ApA is intramolecular interaction between 2′-O-methyl and the flexible O(3′)—P—O(5′) bridge, the interaction causing some perturbation in the magnitudes of ω/ω′, causing destacking. The destacking will lead to an increase in _χCN by a few degrees, causing an increase in ²E populations; the latter in turn will shift the 3′ phosphate group from g^? to g⁺ domains. In short, a coupled series of conformational events is envisioned at the onset of destacking, made feasible by the interaction between the 2′-O-methyl group and the swivel O(3′)—P—O(5′) bridge.     

Synthesis,In Vitro Anti-HIV Activity,and Biological Stability of 5′-O-Myristoyl Analogue Derivatives of 3′-Fluoro-2′,3′-Dideoxythymidine (FLT) as Potential Bifunctional Prodrugs of FLT

Abstract

A group of 5′-O-myristoyl analogue derivatives of FLT (2) were evaluated as potential anti-HIV agents that were designed to serve as prodrugs to FLT. 3′-Fluoro-2′,3′-dideoxy-5′-O-(12-methoxydodecanoyl)thymidine (4) (EC₅₀ = 3.8 nM) and 3′-fluoro-2′,3′-dideoxy-5′-O-(12-azidododecanoyl)thymidine (8) (EC₅₀ = 2.8 nM) were the most effective anti-HIV-1 agents. There was a linear correlation between Log P and HPLC Log retention time for the 5 ′-O-FLT esters. The in vitro enzymatic hydrolysis half-life (t½), among the group of esters (3–8) in porcine liver esterase, rat plasma and rat brain homogenate was longer for 3′-fluoro-2′,3′-dideoxy-5 ′-O-(myristoyl)thymidine (7), with t½ values of 20.3, 4.6 and 17.5 min, respectively.     

Ribosylation of Pyrimidine 2′-Deoxynucleosides

Abstract

The previously developed method for the preparation of 2′-O-D-ribofuranosyl-nucleosides is extended to ribosylation of 2′-deoxynucleosides. The scope and limitations of this reaction are discussed.     

Studies on Synthesis and Structure of O-β-D-Ribofuranosyl(1″→2′)-ribonucleosides and Oligonucleotides♣

Abstract

Minor nucleosides found in several eukaryotic initiator tRNAs_i ^Met, O-β-D-ribofuranosyl(1″→2′)adenosine and -guanosine (Ar and Gr), as well as their pyrimidine analogues, were obtained from N-protected 3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)ribonucleosides and 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose in the presence of tin tetrachloride in 1,2-dichloroethane. A crystal structure has been solved for 2′-O-ribosyluridine. The 3′-phosphoramidites of protected 2′-O-ribosylribonucleosides were prepared as the reagents for 2′-O-ribofuranosyloligonucleotides synthesis. O-β-D-Ribofuranosyl(1″→2′)adenylyl(3′→5′)guanosine (ArpG) was obtained and its structure was analysed by NMR spectroscopy.

     

Synthesis of Methylene-Bridged Analogues of Nicotinamide Riboside,Nicotinamide Mononucleotide and Nicotinamide Adenine Dinucleotide

Abstract

5-O-tert-Butyldimethylsilyl-1,2-O-isopropylidene-3(R)-(nicotinamid-2-ylmethyl)-α-D-ribofuranose (11a) and ?3(R)-(nicotinamid-6-ylmethyl)-α-D-ribofuranose (11b) were prepared by condensation of 5-O-tert-butyldimethylsilyl-1,2-O-isopropylidene-α-D-erythro-3-pentulofuranose (10) with lithiated (LDA) 2-methylnicotinamide and 6-methylnicotinamide, respectively, and then deprotected to give 1,2-O-isopropylidene-3-(R)-(nicotinamid-2-ylmethyl)-α-D-ribofuranose(12a) and 1,2-O-isopropylidene-3(R)-(nicotinamid-6-ylmethyl)-α-D-ribofuranose (12b). Benzoylation as well as phosphorylation of compounds 12 afforded the corresponding 5-O-benzoate (13b) and 5-O-monophosphates (14a and 14b). Treatment of 13b with CF₃COOH/H₂O caused 1,2-de-O-isopropylidenation with simultaneous cyclization to the corresponding methylene-bridged cyclic nucleoside - 3′,6-methylene-1-(5-O-benzoyl-β-D-ribofuranose)-3-carboxamidopyridinium trifluoro-acetate (8b) - restricted to the “anti” conformation. In a similar manner compounds 14a and 14b were converted into conformationally restricted 2,3′-methylene-1-(β-D-ribofuranose)-3-carboxamidopyridinium-5′-monophosphate (9a - “syn”) and 3′,6-methylene-1-(β-D-ribofuranose)-3-carboxamido -pyridinium-5′monophosphate (9b - “anti”) respectively. Coupling of derivatives 12a and 12b with the adenosine 5′-methylenediphosphonate (16) afforded the corresponding dinucleotides 17. Upon acidic 1,2-de-O-isopropylidenation of 17b, the conformationally restricted P¹-[6,3′-methylene-1-(β-D-ribofuranos-5-yl)-3-carboxamidopyridinium]-P²-(adenosin-5′-yl)methylenediphosphonate 18b -“anti” was formed. Compound 18b was found to be unstable. Upon addition of water 18b was converted into the anomeric mixture of acyclic dinucleotides, i. e. P¹-[3(R)-nicotinamid-6-ylmethyl-D-ribofuranos-5-yl]-P²-(adenosin-5′-yl)-methylenediphosphonate (19b). In a similar manner, treatment of 17a with CF₃COOH/H₂O and HPLC purification afforded the corresponding dinucleotide 19a.

     

Structure and Hypotensive Effect of Flavonoid Glycosides in Kinkan (Fortunella japonica) Peelings

An attempt was made to isolate the hypotensive substances from a hot water extract of kinkan. Eight flavonoid glycosides were isolated by repeated chromatography and by gel filtration after extracting with n-butanol and treating with lead subacetate. Their structures were established to be 6,8-di-C-glucosylapigenin (1), 3,6-di-C-glucosylacacetin (2), 2″-O-α-l-rhamnosyl-4′-O-methyl-vitexin (3), 2″-O-α-l-rhamnosyl-4′-O-methylisovitexin (4), 2″-O-α-l-rhamnosylvitexin (5), 2″-O-α-l-rhamnosylorientin (6), 2″-O-α-l-rhamnosyl-4′-O-methylorientin (7) and ponicilin (8) by UV. MS, ¹-NMR and ¹³C-NMR spectroscopy, and by sugar analysis. Each component was intravenously injected in SHR-SP (0.5 ~ 1.0 mg/100 g of body weight), 1, 2, 5 and 6 were found to lower the rat blood pressure.

Among these compounds, 2, 3, 4, 6 and 7 were new flavone glycosides.     

Highly Diastereoselective Synthesis of (2′S)-[2′-2H]-2′-Deoxyribonucleosides from the Corresponding Ribonucleosides

Abstract

The four (2′S)-[2′-²H]-2′-deoxynucleosides (>90 atom % ²H), were synthesized from the corresponding ribonucleosides involving six steps of reactions, i.e., oxidation of their 2′-hydroxyl group, stereoselective reductive deuteration of the resulting 2′-ketonucleoside intermediates with NaB²H₄ in EtOH-H₂O or EtOH, triflation, bromination with LiBr, highly stereoselective Bu₃SnH-Et₃B reduction of the resulting bromide, and, finally, unmasking.     

NMR study of dideoxynucleotides with anti-human immunodeficiency virus (HIV) activity

The molecular structures of 3′-azido-2′,3′-dideoxyribosylthymine 5′-triphosphate (AZTTP), 2′,3′-dideoxyribosylinosine 5′-triphosphate (ddITP), 3′-azido-2′,3′-dideoxyribosylthymine 5′-monophosphate (AZTMP) and 2′,3′-dideoxyribosyladenine 5′-monophosphate (ddAMP) have been studied by NMR to understand their anti-HIV activity. For ddAMP and ddITP, conformations are almost identical with their nucleoside analogues with sugar ring pucker equilibriating between C3′-endo (∼75%) and C2′-endo (∼25%). AZTMP and AZTTP on the other hand show significant variations in the conformational behaviour compared with 3′-azido-2′,3′-dideoxyribo-sylthymine (AZT). The sugar rings for these nucleotides have a much larger population of C2′-endo (∼75%) conformers, like those observed for natural 2′-deoxynucleosides and nucleotides. The major conformers around C5′-O5′, C4′-C5′ and the glycosidic bonds are the β^t, γ⁺ and anti, respectively.     

Simultaneous Removal of Benzyl and Benzyloxycarbonyl Protective Groups in 5′-O-(2-Deoxy-α-D-Glucopyranosyl)Uridine by Catalytic Transfer Hydrogenolysis

Synthesis of N ³,2′,3′-O-tris-(benzyloxycarbonyl)uridine and its use in the synthesis of 5′-O-(2-deoxy-α-d-glucopyranosyl)uridine is described. Simultaneous removal of benzyl and benzyloxycarbonyl groups was accomplished by catalytic transfer hydrogenolysis in the presence of Pearlman's catalyst without competing side reactions.     

Three Antinematodal Diterpenes from Euphorbia kansui

Three compounds, 20-O-acetyl-[3-O-(2′E,4′Z)-decadienoyl]-ingenol (1), 20-O-acetyl-[5-O-(2′E,4′Z)-decadienoyl]-ingenol (2) and 3-O-(2′E,4′Z)-decadienoylingenol (3), were isolated from Euphorbia kansui under the bioassay-guided method. Each compound showed the same antinematodal activity against the nematode, Bursaphelenchus xylophilus, at a minimum effective dose (MED) of 5 μg/cotton ball.     

Synthesis and Conformational Studies of O 5′, 6-Methanouridine—A New Type of Pyrimidine Cyclonucleoside

Abstract

The 5-oxo-6-methylene-pyrimidine-2,4-dione intermediate (6) that is formed when 5-acetoxy-6-acetoxymethyl-1-β-D-(5-O-acetyl-2,3-O-isopropylidene)-ribofuranosyluracil (5) is treated with sodium hydroxide undergoes cyclization at pH 14 to give 2′,3′-O-isopropylidene-5-hydroxy- O ⁵, 6-methanouridine (8) in good yield. Conversion of 8 into the 5-triflate ester 14 followed by reduction with [(Ph)₃P]₄Pd/Bu₃SnH and deblocking with acetic acid then affords O ^5′, 6-methanouridine (4) Conformational studies (NOE difference spectra, vicinal ¹H-¹³C coupling constants, NOESY and CD spectra, molecular modeling) indicate that the C7-methylene group of 4 projects towards the furanose ring oxygen atom, producing a glycosyl rotation angle of about ? 160°.     

Dilignol glycosides from needles of Picea abies

(2R,3R)-2 3-Dihydro-2-(4′-hydroxy-3′-methoxyphenyl)-3-(hydroxymethyl)-7-methoxy-5-benzofuranpropanol 4′-O-β-d-glucopyranoside [dihydrodehydrodiconiferyl alcohol glucoside], (2R,3R)-2 3-dihydro-7-hydroxy-2-(4′-hydroxy-3′-methoxyphenyl)-3-(hydroxymethyl)-5-benzofuranpropanol 4′-O-β-d-glucopyranoside and 4′-O-α-l-rhamnopyranoside, 1-(4′-hydroxy-3′-methoxyphenyl)-2- [2″-hydroxy-4″-(3-hydroxypropyl)phenoxy]-1, 3-propanediol 1-O-β-d-glucopyranoside and 4′-O-β-d-xylopyranoside, 2,3-bis[(4′-hydroxy-3′-methoxyphenyl)-methyl]-1,4-butanediol 1-O-β-d-glucopyranoside [(?)-seco-isolariciresinol glucoside] and (1R,2S,3S)-1,2,3,4-tetrahydro-7-hydroxy-1-(4′-hydroxy-3′-methoxyphenyl)-6-methoxy-2 3-naphthalenedimethanol α²-O-β-d-xylopyranoside [(?)-isolariciresinol xyloside] have been isolated from needles of Picea abies and identified.     

Process Development for the Synthesis of 5′-O-(4,4′-Dimethoxytrityl)-N 2-isobutyryl-2′-O-(2-methoxyethyl)-guanosine—A Potential Precursor for the Second Generation Antisense Oligonucleotides: An Improved Process for the Preparation of 2′-O-Alkyl-2,6-diaminopurine Riboside

Abstract

An efficient four step process for the preparation of 5′-O-(4,4′-dimethoxytrityl)-N ²-isobutyryl-2′-O-(2-methoxyethyl)-guanosine 1 was developed. Direct 2′-O-alkylation of 2,6-diaminopurine riboside 2 was accomplished via inexpensive and commercially available reagents such as KOH, DMSO and alkyl halides at room temperature in 4–6 hrs. Pure 2′-O-(2-methoxyethyl)-DAPR 3 was isolated by crystallization from methanol. Enzymatic deamination of 3 followed by selective N ²-isobutyrylation and 5′-O-dimethoxytritylation furnished desired 1 in high yield and purity. Fully optimized four step synthetic process has been scaled up to the pilot plant level.